Remove and discard s/n without loosing the spheroplasts (better with pipette).

With cut-tip add 1 ml of SOS and gently resuspend by slow pipetting.

Incubate at 30°C without shaking for 30 minutes.

Plate spheroplasts as follows:

A) Standard platting:
1. Invert the tube once and add 5 ml (12 ml if 15 cm plates are used) of molten top agar.
2. Close the tube and mix twice by inversion.
3. Pour onto a 37°C pre-warmed sorb-containing plates
4. Tilt the plate to distribute evenly the agar.
5. Incubate upright for at least 15 min. at room temp.
6. Grow at 30°C for 5-6 days (preventing drying after the 2nd day).

B) Platting facilitating “colony breakthrough”:
1. Invert the tube once and add 10.5 ml (with 10 ml plastic pipette) of molten top agar.
2. Close the tube and mix twice by inversion.
3. With the same pipette used in step 1 distribute the spheroplasts-agar onto a big (15 cm) sorb-containing plates pre-warmed to 50°C.
4. Tilt the plate to distribute evenly the agar (this is critical).
5. Incubate upright for at least 15 min. at room temp.
6. Grow at 30°C for 5-6 days (preventing drying after the 2nd day).